ABSTRACT

This research paper was the results of activity of MSc students of Food Science and Technology, attending the class “Biotechnology of Functional Starter”. Five strains of bifidobacteria (Bifidobacterium animalis subsp. lactis; B. longum subsp. infantis; B. breve; B. animalis subsp. animalis; B. bifidum) were evaluated in order to assess their suitability as functional starter cultures, by studying the following technological and probiotic traits: growth at different temperatures, NaCl amounts and pH values; acidifying ability; metabolism (arginin deamination, esculin hydrolysis, acetoin production); survival at low pH and in presence of bile salts; hydrophobic properties; antibiotic resistance. After laboratory assays and strain selection through a multivariate analyses, it was highlighted that B. longum subsp. infantis and B. animalis subsp. lactis represent a good compromise as potential functional starter cultures, as B. animalis subp. lactis showed a high growth index at pH 5 and good values at 25˚C and 30˚C, as well as the minimal viability loss at pH 2.5. B. longum subsp. infantis DSMZ 20088 was the best microorganism for its growth index in presence of 6.5% of salt added and at 25˚C and 30˚C.

1. Introduction

Bifidobacteria were isolated for the first time in 1899 by Tissier from the stools of breast-fed infants. These Gram-positive and anaerobic microorganisms, not producing any type of gas, were named for their bifurcate morphology (from Latin bifidum) Bacillus bifidus [1].

Then, Orla-Jensen observed that Bacillus bifidus was able to produce lactic acid and classified it in the family of Lactobacteriaceae under the name of Lactobacillus bifidus. Although in 1924 Orla-Jensen introduced the genus Bifidobacterium as a separate taxon, the name L. bifidus remained until 1970. Except for B. dentium (the etiological cause of dental caries), bifidobacteria are regarded as safe [2]. A number of bifidobacteria now have a long history of safe use as dietary adjuncts; B. adolescentis, B. animalis, B. lactis, B. bifidum and B. breve have GRAS (generally regarded as safe) status [1].

Starter cultures are generally designed to assure food safety, shelf-life, technological and economic feasibility criteria. Apart from these traditional properties, new starter cultures should take into account the risks posed by the formation of biogenic amines in food, the development and spreading of bacterial resistance to antibiotics, protection against harmful bacteria either by the production of antimicrobials (bacteriocins) or acidification. The ability of starter cultures to compete with the natural microbiota of raw materials, as well as technological performances, relies upon the ability to survive in the conditions encountered in food (salt, temperature, pH, preservatives).

Probiotics for human use contribute to organoleptic, rheological and nutritional characteristic of foods. They have also a positive effect on the intestinal microflora of the host.

Their optimal characteristics include tolerance to the conditions present in the gastrointestinal tract (resistance to gastric juices, bile), the ability to adhere to the intestinal surface, the production of antimicrobial substances and the ability to modulate the immune response of host [3].

A new kind of starter cultures are the functional starters, i.e. microorganisms acting at the same time as probiotic and starter. Functional starter cultures are used for the improvement of aroma, to obtain safe products because of their ability to produce bacteriocins, for their ability to enrich food matrix with micronutrients [4].

Some reports are available on the use of lactobacilli as functional starter cultures [4]; however, to the best of our knowledge there are no data on bifidobacteria as functional starter microorganisms. Therefore, the main topic of this research was to study the technological and probiotic characteristics of some strains of bifidobacteria; in particular, we focused on B. animalis subsp. lactis; B. longum subsp. infantis; B. breve; B. animalis subsp. Animalis; B. bifidum, assessing:

1) Growth at different temperatures, NaCl amounts and pH values;

2) Acidifying ability;

3) Metabolism (arginin deamination, esculin hydrolysis, acetoin production) ;

4) Survival at low pH and in presence of bile salts;

5) Hydrophobic properties;

6) Antibiotic resistance.

These assays were used as representative of the technological (growth under different conditions, acidification, metabolism) and probiotic abilities (survival at low pH and in presence of bile salts, resistance to antibiotics, hydrophobicity) to assess the suitability of bifidobacteria as multifunctional starter cultures.

2. Materials and Methods

2.1. Strains

This research focused on 5 strains of bifidobacteria purchased from a Public Collection (Deutsche Sammlung von Mikroorganismem und Zellkulturen’s collection, Braunschweig, Germany, DSMZ): B. animalis subsp. lactis DSMZ 10140; B. longum subsp. infantis DSMZ 20088; B. breve DSMZ 20213; B. animalis subsp. Animalis DSMZ 20104; B. bifidum DSMZ 20456. Before each experiment, the microorganisms were grown in MRS broth (Oxoid Milan, Italy), added with 0.5 g/l of cysteine (Sigma-Aldrich, Milan, Italy) (cMRS) and incubated at 37˚C for 48 h under anaerobic conditions, in order to attain a cell concentration of 9 log cfu/ml.

2.2. Metabolism

The following metabolic properties were assessed:

Deamination of arginine. The deamination of arginine was assessed in the substrate of Abd-El-Malek, buffered at pH 7.0. The composition of the substrate is: tryptone (Oxoid) 5 g/l; yeast extract (Oxoid) 2.5 g/l; glucose (C. Erba) 0.5 g/l; K₂HPO₄ (J.T. Baker, Milan) 2 g/l; arginine hydrochloride (Sigma-Aldrich) 3 g/l. The substrate was distributed into sterile test tubes (5 ml), inoculated with ca. 7 log cfu/ml of each strain separately and incubated at 37˚C for 96 h. The test was considered positive if, after the addition of Nessler’s reagent (C. Erba), the samples turned to orange.

Hydrolysis of esculin. This assays was performed using aliquots of 5 ml of MRS broth, buffered at pH 6.5 and supplemented with esculin (2 g/l) (Sigma-Aldrich) and ammonium iron citrate (1 g/l) (C. Erba). The samples were inoculated with ca. 7 log cfu/ml of each strain separately and incubated at 37˚C for at least 72 h. The black colour of the medium after the incubation period denoted hydrolysis of esculin.

Production of acetoin. The production of acetoin was determined on glucose phosphate broth consisting of: bacteriological peptone (Oxoid) 5 g/l; glucose 5 g/l; K₂HPO₄ 5 g/l. After inoculation (7 log cfu/ml), the samples were incubated for 4 - 7 days at 37˚C. The test was considered positive if the colour of the substrate turned to red after the addition of a 6%-solution of α-naphthol (Sigma Aldrich) in ethanol and a 16%-aqueous solution of NaOH.

2.3. Effect of NaCl, pH and Temperature

The assay was performed in cMRS broth, adjusted at pH 5.0 through HCl 1.0 N, or added with different concentrations of NaCl (2-4-6.5%). Otherwise, the effect of the temperature was studied in not-modified cMRS broth (pH 6.0 - 6.2), incubated at 25˚C, 30˚C, 37˚C and 44˚C.

The samples were inoculated with 3 log cfu/ml of each strain separately, and incubated at 37˚C (effect of pH and salt) or at 25˚C - 44˚C (effect of the temperature). Aliquots of not-modified MRS broth, inoculated with the five strains and incubated a 37˚C, were used as controls.

Microbial growth was evaluated after 24, 48 and 96 h through absorbance measurement at 600 nm using a Shimadzu UV-visible spectrophotometer (Shimadzu Europe Ltd., Duisburg, Germany). Data were modeled as Growth Index [5], modified as follows by Bevilacqua et al. [6,7]:

where Abs_s is the absorbance of the samples at different pH values, NaCl concentrations or temperature, and Abs_c the absorbance of the control sample. All the experiments were conducted in duplicate on two independent batches.

2.4. Acidification

Aliquots of MRS broth of 10 ml were inoculated with 6 log cfu/ml of each strain separately and incubated at 30˚C, 37˚C and 44˚C. The acidifying ability was assessed as decrease of the pH of the medium after 24 and 48 h; pH measurements were performed through a pH-meter Crison mod 2001 (Crison Instruments, Barcelona, Spain). All the experiments were conducted in duplicate over two independent batches.

2.5. Survival at 60˚C for 30 min

Microorganisms were grown in cMRS broth, incubated at 37˚C for 48 h; then, cultures were heat-treated at 60˚C for 30 min in a water-bath. After heat-treatment, 100 μl of these cultures were used to inoculate aliquots of 5 ml of cMRS broth, then incubated at 37˚C for 24 h. Microbial growth was evaluated through absorbance measurement at 600 nm. All the experiments were performed in duplicate.

2.6. Survival at pH 2.5 and in Presence of 0.3% of Bile Salts

Aliquots of saline solution (0.9% NaCl), adjusted at pH 2.5 or added with 0.3% of bile salts (Oxoid), were inoculated with ca. 7 log cfu/ml of each strain separately. Then, the samples were incubated at 37˚C for 3 h. Bifidobacteria viability was evaluated through pour plate method on cMRS agar, incubated at 37˚C for 48 h under anaerobic conditions. The analyses were performed on duplicate over two different batches. Aliquots of saline solution at pH 7 and not containing bile salts, but inoculated with bifidobacteria, were used as controls.

Data were modelled as viability loss referred to controls, as follows:

where N_s and N_c were cell counts (log cfu/ml) in the samples acidified at pH 2.5 or added with bile salts and in the control, respectively.

2.7. Antibiotic Resistance

Antibiotic resistance assay was performed through the agar diffusion technique, according to the protocol established by the NCCLS [8]. The strains of bifidobacteria, previously grown in cMRS broth, were streaked on cMRS agar plates through sterile swabs. Then, the disks containing the antibiotics were placed on the surface of the plates.

The plates were incubated at 37˚C for 24 h under anaerobic conditions. At the end of the incubation period, the presence of a halo around the disk of the antibiotic revealed susceptibility of the target towards the antibiotic. All tests were performed in triplicate. The antibiotics were:

1) Ampicillin (33 µg);

2) Vancomycin (70 µg);

3) Erythromycin (78 µg);

4) Gentamicin (40 µg);

5) Streptomycin (100 µg);

6) Chloramphenicol (60 µg);

7) Tetracyclines (80 µg);

8) Ciprofloxacin (10 µg);

9) Trimethoprim (52 µg).

All the antibiotics were purchased from Neo Sensitabs^® (Taastrup, Denmark).

2.8. Hydrophobic Properties

The ability to adhere to intestinal mucosa was evaluated indirectly as hydrophobic property, i.e. as the ability of hyrocarbons (hexadecane) to catch cells. The assay was conducted as follows:

1) 10 ml of cell cultures were centrifuged at 4000 rpm for 10 min;

2) then, the supernatant was discarded and the pellet suspend in 25 ml of PBS (0.8 g/l K₂HPO₄; 0.68 g/l K₂HPO₄; 8.77 g NaCl, acidified at pH 2.0 with HCl 2.0 N (cell suspension);

3) for each strain two different samples were prepared: control (9.5 ml of cell suspension + 0.5 ml of water) and active sample (9.5 ml of cell suspension + 0.5 ml of hexadecane-C. Erba);

4) the samples were shaken for 10 s and left under static conditions for 10 min;

5) the ability of hexadecane to catch cells was evaluated through absorbance measurement at 600 nm after 30, 60, 90 and 120 min.

All the analysis were performed in duplicate and data modelled as hydrophobic index:

where for each time of analysis ΔAbs_s and ΔAbs_c are the decrease in absorbance in the sample containing the hexadecane and in the control, respectively.

2.9. Statistical Analysis

Data were analyzed through one-way analysis of variance (one-way ANOVA) and Tukey’s test through the software Statistica for Windows, ver. 10.0 (Statsoft, Tulsa, Okhla.).

Moreover, the results of technological and probiotic characterization were used as input values to run a Principal Component Analysis through the add-in-soft component of Excel XLSTAT (Addinsoft, Paris, France).

3. Results and Discussion

3.1. Metabolic Traits

Table 1 reports the results for the metabolic characterization of bifidobacteria. All the strains were able to perform the hydrolysis of esculin but not the deamination of arginin; concerning the production of acetoin, this characteristic was recovered only for two strains: B. animalis subsp. lactis DSMZ 10140 and B. animalis subsp. animalis DSMZ 20104. Finally, bifidobacteria did not survive a heat-shock at 60˚C for 30 min.

3.2. Growth under Different Conditions and Acidification

One of the most important trait for the selection of a suitable starter is the study of growth under conditions simulating food matrix for which the starter is intended to. Keeping in mind a possible applications of bifidobacteria as suitable functional starter cultures for dairy products, the technological challenges performed were the growth under acidic conditions, salt resistance and growth in a wide range of temperature.

Figure 1 shows the data of the assays under acidic conditions (pH 5.0) after 24 h of incubation. B. animalis subsp. lactis DSMZ 10140, B. animalis subsp. animalis DSMZ 20104 and B. breve DSMZ 20213 were not affected by the relatively low pH, as one could infer from Growth Index (ca. 95% - 100%); otherwise, B. longum subsp. infantis DSMZ 20088 and B. bifidum DSMZ 20456 were partially inhibited and experienced Growth Indices of 47% and 63%, respectively.